Genetic analysis of spontaneous and 6-N-hydroxylaminopurine and propiolactone induced Adp+ mutants in Saccharomyces yeasts

652 spontaneous and 6-N-hydroxylaminopurine and propiolactone-induced mutants were obtained in yeast. 598 of them were LYS2 mutants. Detailed genetic analysis of the mutants was performed, including analysis of growth pattern on lysineless medium, suppressibility by nonsense suppressors of three types and localization on the recombination map of the LYS2 gene. Mutants induced by different agents were different for all these criteria, except for distribution among the map regions.     

Mutability of LYS2 gene in diploid Saccharomyces yeasts. II. Frequency of mutants induced by 6-N-hydroxylaminopurine and propiolactone

Chemical mutagens 6-N-hydroxylaminopurine (HAP) and propiolactone (PRO) induce Lys2 mutants with high frequency in diploid yeast Saccharomyces cerevisiae. HAP induces such mutants even in tetraploid strains. The genetic analysis of mutants was performed. It is shown that PRO induces mutants by means of "mutation-mitotic segregation" mechanism, while HAP induces mutants through novel mechanism "both allele mutation". Manifestation of such mechanism is the null fertility after meiosis of diploid mutants induced by HAP.     

Yeast resistance to polyene antibiotics. IV. A study of the inheritance of changes in the sterol composition of Saccharomyces cerevisiae yeasts caused by mutations of nystatin resistance

Sterol content in haploid and diploid strains of yeast having mutations of resistance to nystatin were studied by UV spectrometry method. Heterozygous diploids carrying one or two nystatin resistance mutations have, as a rule, the sterol content of the wild type strains. Segregants of the same genotype demonstrate differences in sterol content. Double mutants nys1 nys2 and nys1 nys3 have UV spectra typical for single nys2 and nys3 mutants, respectively. Double mutants nys1 nysX are characterized by a "mixed" UV spectra of sterols.     

Rapid identification of non-allelic nystatin resistance mutations inDictyostelium discoideum

Spontaneous nystatin resistance mutations inDictyostelium discoideum fall into three complementation groups;nys A. nys B andnys C. We demonstrate three methods for rapidly distinguishing mutations in the three complementation groups. In the first methodnys B andnys C mutations are identified by their sensitivity to the sterol biosynthesis inhibitors azasterol A25822B and fenarimol respectively. The second method exploits the differential sensitivities of thenys mutations to the polyene antibiotic pimaricin. In the last method we show thatnys C and non-nys C mutants can be distinguished on the basis of the Lieberman-Burchard color reaction for sterols.     

The effect of mutation him1 characterized by enhanced induced mutagenesis on the genetic effects of 6-N-hydroxylaminopurine in Saccharomyces cerevisiae

The him1 mutation has been shown to influence the genetic effects of the mutagenic purine base analog 6-hydroxylaminopurine, i. e. inactivation of haploid cells, mutation induction, and inhibition of DNA synthesis in vivo. The influence observed is well consistent with the idea that the him1 mutation affects mismatch correction. We present evidence that during in vivo DNA replication 6-hydroxylaminopurine incorporates into the yeast DNA.     

Mutants of Saccharomyces cerevisiae supersensitive to the mutagenic effect of 6-N-hydroxylaminopurine

Yeast mutants hypersensitive to the mutagenic action of 6-N-hydroxylaminopurine (HAP) were obtained by EMS mutagenesis. One of the mutants segregated monogenically and possessed reduced capacity to utilize HAP as a purine source. A set of diploids suitable for parallel study of mutagenesis and induction of recombination, and differing in the trait of mutability after exposure to HAP ("hm" trait or HAP mutability), were constructed. It was shown that a weak recombinogenic effect of HAP is not enhanced in "hm" mutants when HAP mutability increases.     

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.     

Nitrification in vitro by a range of filamentous fungi and yeasts

A wide range of fungi including yeasts, growing on Czapek Dox medium, nitrified added ammonium and the ammonium released by urea hydrolysis. Phanerochaete chrysosporium and Hymenoscyphus ericiae were the only fungi tested which failed to nitrify. A soil yeast (isolate 1) was the most active nitrifier of ammonium in vitro , forming 0·80 μg nitrate mg^-1 biomass over the 7 d incubation period.     

Development of ciprofloxacin resistance due to chromosomal mutations induced by 2-nitrofluorene

In this paper we investigated the ability of 2-nitrofluorene to induce mutations leading to antibiotic resistance in quinolone-sensitive strain Salmonella typhimurium. After preincubation of bacteria with 2-nitrofluorene, the frequency of mutation to ciprofloxacin resistance was 57 fold higher than in the case of spontaneous mutability. Some of resultant resistant colonies showed a great increase of ciprofloxacin MIC.     

Species range expansion by beneficial mutations

A species' range can be limited when there is no genetic variation for a trait that allows for adaptation to more extreme environments. We study how range expansion occurs by the establishment of a new mutation that affects a quantitative trait in a spatially continuous population. The optimal phenotype for the trait varies linearly in space. The survival probabilities of new mutations affecting the trait are found by simulation. Shallow environmental gradients favour mutations that arise nearer to the range margin and that have smaller phenotypic effects than do steep gradients. Mutations that become established in shallow environmental gradients typically result in proportionally larger range expansions than those that establish in steep gradients. Mutations that become established in populations with high maximum growth rates tend to originate nearer to the range edge and to cause relatively smaller range expansion than mutations that establish in populations with low maximum growth rates. Under plausible parameter values, mutations that allow for range expansion tend to have large phenotypic effects (more than one phenotypic standard deviation) and cause substantial range expansions (15% or more). Sexual reproduction allows for larger range expansions and adaptation to more extreme environments than asexual reproduction.     

The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin

The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membrane’s molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membrane's expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin.     

Kinetics of apparent cell death in yeasts induced by ethanol

Summary Evidence is presented that adaptation of yeast cells to ethanol results in a reduced loss of cell viability induced by exposure to that agent. In line with earlier work, an exponential model is shown to apply when the concentration of ethanol exceeds a critical value, beyond which cell growth cannot occur. Such an exponential model is consistent with the absolute theory of reaction rates. Adaptation of yeast cells to 7% w/v ethanol lowers the specific rate of cell death at various ethanol concentrations by a factor of some 40 fold compared to a non-adapted culture.     

Incidence of yeasts and influence of nystatin on their control in a group of burned children

The incidence of yeasts in the oral cavity, rectum and urine of a population of 60 children hospitalized for treatment of acute second and third degree burns was approximately the same at the time of their admission as would be expected in healthy subjects. After hospitalization, the incidence of yeasts was reduced in the intestinal tract of acute patients who received nystatin orally but increased in the oral cavity. The majority of 418 yeasts were inhibited in vitro by less than 50 units/ml nystatin and only 6 yeasts were resistant to more than 3.1 g/ml amphotericin B. The oral cavity appeared to act as a significant reservoir from which yeasts spread to cause or contribute to the deaths of 2 of 5 patients who died during the study.     

Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the LYS2 gene of yeast Saccharomyces cerevisiae

We used the LYS2 gene mutational system to study mutation specificity of the base analog 6-N-hydroxylaminopurine (HAP) in yeast. We characterized phenotypes of mutations using codon-specific nonsense suppressors and the test employing inactivation of the release factor Sup35 due to overexpression and formation of prion-like derivative [PSI]. We have shown that HAP induces predominantly nonsense mutations. While the tests using codon-specific nonsense-suppressors allowed to identify only about 50% of nonsense-mutations, all the nonsense-mutations were identified in the test with defective Sup35. We determined and analyzed the spectrum of HAP-induced nucleotide changes in two regions of the gene. HAP induces predominantly GC-->AT transitions in a hotspots of a central position of trinucleotide GGA or AGG. Directionality of these transitions is consistent with the idea that initial dHAPMP incorporation in the leading strand is more genetically dangerous than in lagging DNA strand. We revealed a specific context inhibitory for HAP mutagenesis, a "T" in -1 position to mutation site.     

HIV‐1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations

Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Ω‐shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir‐resistant viruses. Copyright © 2009 John Wiley & Sons, Ltd.     

Mitochondrial DNA exhibits resistance to induced point and deletion mutations

The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed Muta^TMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure.